1. Field of the Invention
The invention relates to novel methods for generating and screening peptides which are useful for a variety of applications.
2. Description of the Background Art
Small peptides (2-15 amino acids) are capable of possessing unique spatial and thermodynamic properties which allow them to bind specifically to particular proteins. The binding of these peptides to such proteins can transiently or permanently eliminate or activate the function of those proteins. Thus a peptide which binds to, and eliminates the function of a coat protein of a virus could serve as an effective pharmacologic agent against that virus. Similarly, a peptide which binds to and activates a receptor for taste or olfaction could serve as a useful active agent for flavoring or perfumes. There is considerable interest in the pharmaceutical industry in the development of such useful peptides. The major difficulty is that the production and screening of individual peptides is inefficient and labor intensive. Peptides must be made individually and tested for their ability to bind the protein of interest. Because of the difficulty of making and screening each individual peptide, it is not possible to screen large numbers of peptides. There is, therefore, a need for methods which allow for rapid and efficient screening of a large number of peptides. An ideal system would provide for the simultaneous synthesis and screening of every possible peptide of a particular size.
A variety of proteolytic enzymes, including trypsin, chymotrypsin, pepsin, papain, bromelain, thermolysin and S. griseus proteinase, hydrolyze peptide bonds. The rate of hydrolysis is influenced by temperature, substrate and enzyme concentration, enzyme specificity, and substrate sequence. S. griseus proteinase, papain, and the subtilisin enzymes are known to have broad substrate specificity, but for total enzymatic hydrolysis, the use of a mixture of enzymes is preferred. Hill, "Hydrolysis of Proteins", in Advances in Protein Chemistry, 20: 37, 89-90 (Anfinsen, Jr., et al., eds., 1965).
In 1937, it was demonstrated that a proteolytic reaction could be reversed. Soon thereafter, proteases were used for both stepwise and fragment condensation synthesis of peptides of predetermined sequence. Since the enzymes differed in terms of which peptide bonds would be formed, several different enzymes had to be used in succession to make oligopeptides. Unfortunately, enzymatic chain elongation could endanger preexisting bonds. See Sakina, et al., Int. J. Peptide Protein Res., 31: 245-52 (1988); Kullman, Proc. Nat. Acad. Sci. (U.S.A) 79: 2840-44 (1982).
One problem in enzymatic synthesis of peptides is that hydrolysis is thermodynamically favored under normal conditions. Thus, the equilibrium must be shifted. Homandberg, et al., Biochemistry, 21: 3385-89 (1982) teaches that one may use a "molecular trap", that is, a molecule which has an affinity for a particular peptide of known amino acid sequence, to shift the equilibrium to favor the synthesis of such a peptide.
Varied approaches have been utilized for the purpose of generating diverse populations of peptides.
"Semisynthetic" peptides and proteins have been prepared by (1) limited proteolysis of naturally occurring polypeptides to yield a workable set of fragments, (2) chemical synthesis of an additional oligopeptide, and (3) reconstruction of synthetic and native partners. The technique is typically used to prepare analogues of naturally occurring polypeptides. Chaiken, CRC Critical Reviews in Biochemistry, 255 (Sept. 1981). Ruggeri, et al., P.N.A.S. (U.S.A.), 83: 5708-12 (Aug. 1986) prepared a series of synthetic peptides in lengths up to 16 residues that were modeled on various platelet-binding peptides. The technique used was one of solid state synthesis by chemical means, but using individual compartmentalized peptide resins to impart the desired variety. See Houghten, et al., P.N.A.S. (U.S.A.), 82: 5131-35 (1985). Yet another approach to generating a variety of peptides is through expression of mixed oligonucleotides. See Goff, et al., DNA, 6: 381-88 (1987). None of these approaches involve a balanced equilibrium between random synthesis and random degradation within a large population of peptides. Instead, a known peptide is cleaved at a known site, a known amino acid or peptide fragment is conjugated to the first peptide at that cleavage site, and so forth. What is needed is such a scrambling system, wherein a diverse population of peptides is sampled for binding activity and selective removal of a particular peptide having a desired binding activity but a then-unknown amino acid sequence will result in net selective synthesis of that product without net synthesis of large amounts of every possible product.
The primary use for proteolytic enzymes as catalysts for peptide synthesis has previously been directed toward the synthesis of single, known peptide species. Several patents relate to such enzymatic synthesis of non-random peptides and, to this end, disclose and claim use of protective groups to prevent formation of other peptides. Isowa, U.S. Pat. No. 3,977,773 (1976) relates to use of pepsin in the enzymatic production of peptides containing three or more amino acids. Certain constraints are set on the sequence of the peptide. N- and C-terminal protective groups are broadly recited. Isowa, U.S. Pat. No. 4,086,136 (1978) claims the use of a thiol proteinase (e.g., papain) or a serine proteinase (e.g., subtilisin) in the enzymatic synthesis of peptides containing two or more amino acids. The claim requires that one amino acid or peptide carry an amino protective group and the other a carboxyl protective group, and the latter be one of the following: tertiary alkoxy, benzyloxy, benzylamino, and benzylhydrylamino. Isowa, U.S. Pat. No. 4,116,768 (1978) is drawn to production of a peptide under the action of a metalloproteinase enzyme. N- and C-terminal protective groups are broadly recited. See also Isowa, U.S. Pat. No. 4,119,493 (1978). Isowa, U.S. Pat. No. 4,165,311 (1979) is essentially directed to addition compounds for use in the production of alpha-L-aspartyl-n-phenylalanine alkyl esters (i.e., aspartame-like compounds). Isowa, U.S. Pat. No. 4,256,836 (1981) is drawn to the process for the production of these compounds using a protease. See also U.S. Pat. No. 4,521,514 (1985).
Johansen, U.S. Pat. No. 4,339,534 (1982; De Forened Bryggerier A/S) is broadly directed to the use of an L-specific serine or thiol carboxypeptidase, such as yeast carboxypeptidase, for enzymatic synthesis of peptides. See also Johansen, U.S. Pat. No. 4,806,473. Oyama, U.S. Pat. No. 4,521,514 (1985; Toyo Soda) is directed to a process for recovering protease. Snellman, U.S. Pat. No. 3,544,426 (1965) relates to synthesis of peptide chains by reaction of a carboxylic acid ester, an amine (including a peptide), a phosphoric derivative of a nucleoside, and an enzyme. Morihara, U.S. Pat. Nos. 4,320,197 and 4,320,196 relate to a semisynthesis of human insulin.
Of course, the use of proteolytic enzymes to generate peptides of exclusively pre-existing amino acid sequences through simple degradation of existing proteins is well known. For example, Matsukawa, U.S. Pat. No. 3,855,196 (issued Dec. 17, 1974 to the inventors) decomposes the skeletal muscles of a cervoidae (a taxon including reindeer and caribou) with a protease into low molecular weight peptides with a molecular weight of less than 1,000. These peptides are separated from the crude digest using a gel-type molecular sieve.
Maubois, U.S. Pat. No. 3,993,636 (issued Nov. 23, 1976 to the Institute National de la Recherche Agronomique) ultrafiltered vegetable proteins using membranes filtered with molecular weight cutoffs as low as 2,000 (col. 4, lines 38-44).
Fujimaki, U.S. Pat. No. 3,813,327 (issued Apr. 9, 1974, now expired) claims obtaining an oligopeptide of MW 1,200-2,000 by hydrolyzing a protein with, e.g., subtilisin.
Pieczenik, WO87/01374 further uses such uniform-sized, but variably sequenced peptides derived from simple degradation of existing proteins to identify the recognition sites of antibodies. Alternatively, variable peptides are produced through chemical synthesis from amino acids, or through insertion of fractionated DNA into an expression vector and subsequent expression thereof. Pieczenik does not, however, recognize the advantages of combining random degradation with random synthesis within a chemical system wherein they are in a balanced equilibrium, and disturbing that equilibrium only to favor synthesis of a peptide species that exhibits a desired binding activity.
Binding of peptides by macromolecules in the presence or absence of semi-permeable membranes has previously been described for a variety of purposes. Receptor binding assays are well known in the art, and the screening of peptides for ability to bind to a receptor is conventional. See, e.g., Ruggeri, supra. Westman U.S. Pat. No. 3,649,457 (issued Mar. 14, 1972 to Monsanto Co.) discloses placing a soluble enzyme-polymer conjugate in an enzymatic reaction chamber having a semipermeable wall which permits passage of the enzymatic reaction products but not of the enzyme reagent. In the example, the membrane had an exclusion limit of as low as 1000 (col. 3, lines 15-18). Papain was among the enzymes suggested for use in this system (col. 9, line 48). While the reaction product must be of lower molecular weight than the enzyme-polymer conjugate, "it may be of a higher molecular weight than starting substrate, as when substrate comprises a plurality of reactants which are enzymatically reacted together to give a higher molecular weight product." (col. 13, lines 60-63). Likhite, U.S. Pat. No. 3,843,444 (issued Oct. 23, 1974) discloses a membrane separation process in which a ligand in one medium is attracted across a barrier film to a receptor in a second medium. It must be emphasized that in Likhite's system, neither the receptor nor the ligand actually crosses the barrier; they are selectively collected on opposite sides thereof. (See, e.g., col. 2, lines 20-21, 25-28 and 67-70).
The present invention is directed toward satisfying the need for the generation of large, random populations of peptides of a particular size range and to concomitant selection of potentially useful peptides from such a population and synthesis of analyzable quantities of such peptides without the net production of large quantities of extraneous, random peptides. The invention fills this need by the use of scrambling reactions, i.e., a system at steady state, and preferably at a global equilibrium, between synthesis and degradation of peptides catalyzed by proteolytic enzymes, and by coupling such scrambling reactions to a means for selection and net synthesis of potentially useful peptides through perturbation of the equilibrium, in particular by binding of such peptides by a macromolecule, thus removing such peptides from the system and shifting the equilibrium to favor formation of such bound peptides. Genetically engineered proteins having enzymatic activity are also useful in promoting scrambling reactions and providing random distributions of peptides.